URG4 overexpression is correlated with cervical cancer progression and poor prognosis in patients with early-stage cervical cancer

Upregulator of cell proliferation 4 (URG4) has been implicated in the oncogenesis of certain cancers. However, the correlation between URG4 expression and clinicopathological significance in human cancer remains unclear. Therefore, this study investigated its expression and clinicopathological significance in cervical cancer patients.

R E S E A R C H A R T I C L E Open Access

URG4 overexpression is correlated with cervical cancer progression and poor prognosis in patients with early-stage cervical cancer

Lan Zhang1,2, He Huang2, Longjuan Zhang3, Teng Hou2, Shu Wu1, Qidan Huang2, Libing Song1*and Jihong Liu2*

Abstract

Background: Upregulator of cell proliferation 4 (URG4) has been implicated in the oncogenesis of certain cancers However, the correlation between URG4 expression and clinicopathological significance in human cancer remains unclear Therefore, this study investigated its expression and clinicopathological significance in cervical cancer patients Methods: URG4 expression was examined using quantitative PCR (qPCR) and western blotting in normal cervical epithelial cells, cervical cancer cells, and eight matched pairs of cervical cancer tissues and adjacent noncancerous tissues from the same patient In addition, immunohistochemistry (IHC) was used to examine URG4 expression in paraffin-embedded tissues from 167 cervical cancer patients (FIGO stages Ib1-IIa2) Statistical analyses were performed

to evaluate associations between URG4 expression and prognostic and diagnostic factors

Results: URG4 was significantly upregulated in the cervical cancer cell lines and tissues compared with the normal cells and adjacent noncancerous cervical tissues IHC revealed high URG4 expression in 59 out of the 167 (35.13%) cervical cancer specimens Its expression was significantly correlated with clinical stage (P < 0.0001), tumour size (P = 0.012),

T classification (P = 0.023), lymph node metastasis (P = 0.001) and vaginal involvement (P = 0.002) Patients with high URG4 expression, particularly those who received concurrent chemotherapy and radiotherapy (P < 0.0001), showed a shorter overall survival (OS) and disease-free survival (DFS) compared to those with the low expression of this protein Multivariate analysis revealed that URG4 expression is an independent prognostic factor for cervical cancer patients

Conclusions: Our results demonstrated that elevated URG4 protein expression is associated with a poor outcome

in patients with early-stage cervical cancer URG4 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer

Keywords: URG4, Cervical cancer, Prognosis, Concurrent chemotherapy and radiotherapy, Biomarker

Background

Cervical cancer is the third most commonly diagnosed

gynaecological cancer and the fourth leading cause of

gynaecological cancer deaths worldwide, and it accounted

for 9% (529,800) of the total new cancer cases and 8%

(275,100) of the total cancer deaths among females in

2008 More than 85% of these cases and deaths occurred

in developing countries, including China [1] In China, there are approximately 130,000 new cases and 50,000 deaths due to cervical cancer per year [2] Although the incidence and mortality of cervical cancer have shown downward trends, it is still the major cause of gynaecologic oncology-related death in developing countries, and it is a public health problem worldwide [1] The treatment strat-egy for cervical cancer depends on the clinical stage, which

is defined by the International Federation of Gynaecology and Obstetrics (FIGO) staging system There are several traditional clinical variables that play important roles in the FIGO staging system and patient prognosis, including lymph node metastasis, tumour size and parametrial

* Correspondence: lb.song1@gmail.com; liujih@mail.sysu.edu.cn

1 Sun Yat-sen University Cancer Centre, State Key Laboratory of Oncology in

South China, Collaborative Innovation Centre for Cancer Medicine,

Guangzhou 510060, PR China

2

Department of Gynaecology Oncology, Sun Yat-Sen University Cancer

Centre, Guangzhou 510060, PR China

Full list of author information is available at the end of the article

© 2014 Zhang et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,

involvement [3,4] After surgery, patients with one or

more of the abovementioned clinical variables require

supplementary therapy However, traditional pathological

variables are not sufficiently reliable for predicting clinical

outcomes or for guiding optimal treatment strategies

Many genes, such as annexin A2, Sam68 and HDAC10

[5-7], have been reported to be potentially useful

prog-nostic markers in cervical cancer, but there is still an urgent

need for additional research to identify novel biomarkers to

supply practical information for patient prognosis and

suitable therapeutic options

Upregulated gene 4 (URG4) has been identified as an

oncogene with a full-length mRNA of 3.607 kb that

en-codes a protein of approximately 104 kDa in size This gene

may be associated with the onset of oncogenesis and

cell cycle regulation [8-10] URG4 was initially identified

using subtractive hybridisation in hepatocellular carcinoma

(HCC) cells [8] Previous research on HCC and gastric

cancer evaluating tissue culture and tumour formation

in nude mice has demonstrated that URG4 promotes

HepG2 and GES-1 cell growth and that it is associated

with poor survival In addition, elevated URG4 expression

in HCC and gastric cancer cells leads to upregulation of

cyclin D1, whereas low URG4 expression downregulates

the expression of this gene A study by Chan Xie has

indi-cated that URG4 regulates cyclin D1 expression via the

Akt/FOXO3 signalling pathway by mediating its

prolifera-tive effects on HCC cells [10] In addition, some studies

have shown that the URG4 expression is increased in

different types of cancers [11-13] However, the clinical

significance of this gene in human cervical cancer remains

unknown

In the present study, we demonstrated that the

expres-sion of URG4 is upregulated in cervical cancer cells and

surgical specimens Moreover, its expression in cervical

cancer is associated with clinical stage, tumour size, T

classification, N classification and vaginal involvement

Multivariate analysis revealed that URG4 may be an

inde-pendent biomarker for predicting cervical cancer prognosis

More importantly, its upregulation is indicative of a poor

prognosis, particularly in patients receiving concurrent

chemotherapy and radiotherapy Our results suggest that

URG4 may be an independent biomarker for prognosis

and that it represents a therapeutic target for the

treat-ment of cervical cancer

Methods

Cell lines

A primary culture of normal cervical epithelial cells was

established from a biopsy of noncancerous cervical

epi-thelium and was cultured in complete KeratinocyteSFM

medium (Invitrogen, Carlsbad, CA, USA) Patient consent

was obtained prior to the use of the clinical materials for

research purposes, and the patient consent and protocol

were approved by Sun Yat-sen University Cancer Center Institutional Review Board Eight human cervical cancer cell lines were purchased from the American Type Culture Collection (HeLa, HeLa 229, C-33A, MS751, SiHa and Ca Ski) and the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (HCC 94 and ME-180) The HeLa, HeLa 229, C-33A, MS751 and SiHa cells were cultured in Eagle’s minimum essential medium (Gibco BRL, Rockville, MD) The Ca Ski and HCC 94 cells were cultured

in RPMI-1640 medium (Gibco BRL, Rockville, MD), and the ME-180 cells were cultured in McCoy’s 5A medium (Sigma) supplemented with 10% foetal bovine serum (FBS) (HyClone, Logan, UT, USA)

Samples and clinical characteristics

This study was conducted on a total of 167 paraffin-embedded cervical cancer samples, which were histo-pathologically and clinically diagnosed at the Sun Yat-Sen University Cancer Centre between 1999 and 2005 The clinical and clinicopathological classifications and staging were determined according to the 2009 FIGO criteria All

of the patients enrolled in this study were only found to possess gynaecological tumour(s), and they were treated without preoperative radiotherapy, chemotherapy, or hormonal therapy Patient consent was obtained prior to the use of the clinical materials for research purposes, and the patient consent and protocol were approved by Sun Yat-sen University Cancer Center Institutional Review Board Clinical information pertaining to the samples is summarised in Table 1 The follow-up time for the primary cervical cancer cohort ranged from 0.8 to 187 months, and the median follow-up time was 64.06 months The per-centage of tumour purity in the sections adjacent to the tumours and the normal cervical tissues used for RNA extraction were estimated during routine histopatho-logical analyses

qPCR

Total RNA samples from the cell lines and primary tumour materials were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s in-structions The extracted RNA was pretreated with RNase-free DNase, and 2μg of RNA was used for cDNA synthesis using random hexamers The URG4 sense primer was 5′-CGCAATCATCTCCTTCCATT-3′, and the antisense primer was 5′-GATTTGGGAGAAGTAGCCCC-3′ For the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the sense primer 5′-AATGAAGGGGTCATTGAT GG-3′ and the antisense primer 5′-AAGGTGAAGGTC GGAGTCAA-3′ were used The initial PCR was performed

as follows: denaturation at 95°C for 10 min, followed by

44 cycles of denaturation at 95°C for 15 s, primer annealing

at 60°C for 60 min, and a primer extension step at 65°C for

5 s Upon the completion of these steps, a final extension at

95°C was performed before the reaction mixture was stored

at 4°C qPCR was then conducted to determine the fold increase of URG4 mRNA in each of the primary cervical tumours relative to paired adjacent noncancerous tissue taken from the same patient The primers were de-signed using Primer Express v2.0 (Applied Biosystems) The expression data were normalised to the geometric mean of GAPDH expression to control for variability in expression levels, and all of the experiments were performed in triplicate

Western blotting

Cells at 70% to 80% confluence were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice in radioimmunoprecipitation assay buffer (RIPA; Cell Signaling Technology, Danvers, MA) containing complete protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany) Fresh tissue samples were ground

to powder in liquid nitrogen and lysed using SDS-PAGE sample buffer Equal concentrations of each protein sam-ple (20μg) were separated on 6% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) branes (Immobilon P, Millipore, Bedford, MA) The mem-branes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room

Table 1 Clinicopathological characteristics and URG4

expression in the cervical cancer patients

Number of cases (%) Age, years

Squamous cell carcinoma antigen, ng/ml

FIGO stage

Tumour size, cm

Histological type

Histological differentiation

Deep stromal invasion

Lymphovascular space involvement

Positive parametrium

Positive surgical margin

Vaginal involvement

T classification

Table 1 Clinicopathological characteristics and URG4 expression in the cervical cancer patients (Continued)

N classification

M classification

Concurrent chemotherapy and radiotherapy

Radiotherapy

Recurrence

Vital status (at follow-up)

URG4 expression

0)

temperature The membranes were then incubated with

anti-upregulator of cell proliferation 4 antibody (1:1000,

Sigma, HPA020134) overnight at 4°C URG4 expression

was determined using a horseradish peroxidase-conjugated

anti-rabbit IgG antibody (1:3000, Santa Cruz, SC-2004)

and enhanced chemiluminescence (Pierce) according

to the manufacturer’s protocols The membranes were

probed with anti-α-tubulin mouse monoclonal antibody

(1:1000, Sigma, T5168) as a loading control

Immunohistochemical analysis

Immunohistochemical analysis was performed to assess

alterations in protein expression in the 167 human

cer-vical cancer tissues Briefly, paraffin-embedded specimens

were cut into 4-μm thick sections and baked at 65°C for

30 min The sections were deparaffinised with xylenes and

rehydrated They were then submerged into EDTA

anti-genic retrieval buffer and microwaved for antianti-genic

re-trieval Next, they were treated with 3% hydrogen peroxide

in methanol to quench endogenous peroxidase activity,

followed by incubation with 1% bovine serum albumin to

block any nonspecific binding The sections were then

incubated with an anti-URG4 rabbit polyclonal antibody

(1:150, Sigma, HPA020134) overnight at 4°C Normal goat

serum was used as a negative control After washing with

PBST, the tissue sections were incubated with a biotinyl-ated anti-rabbit secondary antibody (Sigma), followed by further incubation with streptavidin-horseradish peroxid-ase complex (Sigma) The tissue sections were immersed

in 3-amino-9-ethylcarbazole, counterstained with 10% Mayer’s haematoxylin, dehydrated and mounted in Crystal Mount

The degree of immunostaining of the formalin-fixed, paraffin-embedded sections was evaluated independently

by two observers who were blinded to the histopatho-logical features of the samples and the patient data The scores assigned by the two independent investigators were averaged, and they were based on both the proportion of positively stained tumour cells and the intensity of stain-ing The proportion of tumour cells was scored as follows:

1 (<10% positive tumour cells), 2 (10-50% positive tumour cells), 3 (50-75% positive tumour cells), and 4 (>75% positive tumour cells) Staining intensity was graded ac-cording to the following criteria: 0 (no staining); 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown) The staining index was calculated as the product of the proportion

of positive cells and the staining intensity score Using this method of assessment, we evaluated URG4 expression

in cervical cancer cells via a staining index (scored as 0, 1,

Figure 1 Overexpression of URG4 mRNA and protein in cervical cancer cell lines (a and b) Expression levels of URG4 mRNA and protein in cervical cancer cell lines (HeLa, HeLa 229, HCC 94, C33a, Ca Ski, MS751, ME-180 and SiHa) and normal cervical cell lines were examined via western blotting (a) and qPCR (b) The expression levels were normalised against α-tubulin and GAPDH, respectively The error bars represent the standard deviation of the mean (SD), which was calculated from three parallel experiments *P < 0.01.

2, 3, 4, 6, 8, 9 or 12) The cut-off values for URG4

expres-sion were chosen based on a measure of heterogeneity

using the log-rank test with respect to overall survival

(OS) The optimal cut-off values were assigned as follows:

staining scores of≥6 described tumours with high URG4

expression, and scores of≤4 were assigned to those with

low URG4 expression

Statistical analysis

The OS rate was the primary endpoint of this study, and

the secondary endpoint was the disease-free survival

(DFS) of the cervical cancer patients OS was defined as

the duration from the date of each patient’s hospitalisa-tion to the date of death from any cause or to the cen-soring of the patient at the date of the last follow-up DFS was defined as the time from hospitalisation to local, regional, or distant treatment failure, other second primary cancer, or death without evidence of a cervical or second primary cancer

All statistical analyses were conducted using SPSS 11.0 statistical software The relationships between URG4 ex-pression and the clinicopathological characteristics were analysed using the chi-square test and Fisher’s exact test Bivariate correlations between the study variables were

Figure 2 Overexpression of URG4 mRNA and protein in cervical cancer tissues (a) Representative images of western blotting analyses of URG4 protein expression in eight matched pairs of cervical cancer tissues (T) and adjacent noncancerous tissues (ANT) α-Tubulin was used as the loading control (b) The average T/ANT ratios of URG4 mRNA expression in the paired cervical cancer (T) and adjacent noncancerous tissues (ANT) were quantified using qPCR and normalised against GAPDH The error bars represent the standard deviation of the mean (SD), which was calculated from three parallel experiments (c) Immunohistochemical analysis of URG4 protein expression in eight pairs of matched cervical cancer tissues *P < 0.05.

**P < 0.01.

calculated using Spearman’s rank correlation coefficients.

Survival curves were plotted using the Kaplan-Meier

method and were compared using the log-rank test The

clinicopathological characteristics, which are used

exten-sively to predict prognosis in clinical practice, were

eval-uated using univariate and multivariate analysis with the

of <0.05 was considered to be statistically significant

Results

URG4 is overexpressed in cervical cancer cell lines

To evaluate URG4 protein and mRNA expression in

cer-vical cancer cell lines, we used western blotting and qPCR,

and eight cervical cancer cell lines were assessed (HeLa,

HeLa 229, HCC 94, C-33A, Ca Ski, MS751, ME-180 and

SiHa) and compared with a normal cervical epithelial cell

line (N) The URG4 protein was highly expressed in the

cervical cancer cell lines and only weakly expressed in N

(Figure 1a) URG4 mRNA expression was at least 4.5-fold

greater in the cervical cancer cell lines compared to N

(Figure 1b)

URG4 is overexpressed in cervical cancer tissues

To determine whether URG4 is also highly expressed in human cervical cancer clinical samples, we performed qPCR and western blotting analyses on eight cervical tumour samples (T) that were matched with adjacent noncancerous tissue samples (ANT) As illustrated in Figure 2b, URG4 mRNA expression increased by

8.4-to 26.4-fold in all cervical cancer tissues compared 8.4-to the matched adjacent noncancerous tissues Consistent with these data, the URG4 protein was also upregulated in the cervical cancer tissues compared to the surrounding non-tumour regions (Figure 2b)

URG4 overexpression is associated with clinical features

of cervical cancer

We investigated URG4 expression in 167 paraffin-embedded archived cervical cancer tissues using im-munohistochemical staining The samples included 68 stage Ib1 tumours, 59 stage Ib2 tumours, 38 stage IIa1 tumours and two stage IIa2 tumours Among the 167 samples, high levels of URG4 protein expression were detected in 59 (35.5%), and weak or no staining was

Figure 3 Expression of the URG4 protein in cervical cancer tissues from patients at different clinical stages (a) Representative images from immunohistochemical analyses of URG4 expression in normal cervical tissues and cervical cancer tissues at different clinical stages (b) The statistical analyses of the average mean optical density (MOD) of URG4 staining in normal cervical tissues and cervical cancer specimens at different clinical stages (c) The statistical analyses of the average MOD of URG4 staining in the lymph node metastasis group and the lymph node metastasis-free group *P < 0.05.

observed in the remaining 108 (64.7%, Table 1) The

positive rate increased with increasing FIGO stage as

follows: 16.2% for Ib1 (11/68), 45.8% for Ib2 (27/59),

50% for IIa1 (19/38) and 100% for IIa2 (2/2) URG4 was

primarily localised to the plasma membrane (Figure 2)

Furthermore, IHC staining showed that URG4 expression

in the cervical cancer increased with increasing clinical

stage (Figure 3a) Quantitative IHC analysis revealed that the mean optical density (MOD) values of URG4 staining

in all of the cervical cancer samples were higher than those in the normal control cervical tissues In addition, the MOD values of URG4 staining significantly increased with progression from stage Ib1 to IIa2 (P < 0.0001, Figure 3b) Taken together, these observations indicate

Table 2 Correlation of clinicopathological characteristics and URG4 expression in cervical cancer patients

(n = 167)

test P-value Fishertest P-value’s exact Low expression High expression

that high levels of URG4 expression are associated with

the clinical development of early-stage cervical cancer

We further analysed the correlation between URG4

expression and the clinicopathological characteristics of

the patients (Table 1) As summarised in Table 2, there

were no significant correlations between URG4 protein

expression and patient age, M classification, histological

differentiation, SCC expression, histological type, deep

stromal invasion, lymphovascular space involvement,

posi-tive parametrium or posiposi-tive surgical margin in the patients

with cervical cancer However, URG4 expression was

mark-edly associated with clinical stage (P < 0.001), T

classifica-tion (P = 0.003), tumour size (P = 0.012), N classificaclassifica-tion

(P = 0.001) and vaginal involvement (P = 0.004) These

data were further confirmed by Spearman’s correlation

analysis As shown in Table 3, the correlations between

URG4 expression and clinical stage, T classification, N

classification and vaginal involvement were 0.327 (P <

0.0001), 0.250 (P = 0.001), 0.254 (P = 0.001) and 0.236

(P = 0.002), respectively Moreover, the MOD values of

URG4 staining were markedly higher in the lymph node

metastasis group than in the lymph node

metastasis-free group (P < 0.001, Figure 3c)

Taken together, the expression of the URG4 protein is

positively correlated with clinical stage, tumour size, T

classification, N classification and vaginal involvement

Association between URG4 expression and patient

survival

Patient survival analysis showed a clear negative

correl-ation between URG4 protein expression and both the

OS and DFS of cervical cancer patients (bothP < 0.0001,

Figure 4a, b) The cumulative OS and DFS rates for the

patients with high levels of URG4 expression were 52.5%

and 55.9%, respectively, whereas the rates were 97.1% and

89.8%, respectively, for the patients with low or no URG4

expression Moreover, we analysed the prognostic value of

URG4 expression in select patient subgroups that were

stratified according to clinical stage, N classification,

con-current chemotherapy and radiotherapy and radiotherapy

(RT) Patients with tumours exhibiting high URG4

expres-sion had a significantly shorter OS compared to those with

low-URG4-expressing tumours in the Ib1-Ib2 subgroup

(log-rank test, P < 0.0001, Figure 4c), in those without lymph node metastasis (log-rank test,P < 0.0001, Figure 4d) and in those receiving concurrent chemotherapy and radiotherapy (log-rank test, P < 0.0001, Figure 4e) This was not the case for the IIa1-IIa2 subgroup (log-rank test,

P = 0.485, Additional file 1: Figure S1a), the lymph node metastasis group (log-rank test, P = 0.280, Additional file 1: Figure S1b) or the radiotherapy group (log-rank test,

P = 0.06, Figure 4f)

In addition, Cox regression analysis revealed that URG4 expression and deep stromal invasion were independent prognostic factors for poor OS in the cervical cancer patients (Table 4)

Discussion

This is the first study to describe the association of URG4 upregulation with poor prognosis in early-stage cervical cancer patients More importantly, high URG4 expression reduced the OS and DFS of these patients, particularly those who received concurrent chemotherapy and radiotherapy Considering these findings, we suggest that URG4 is a potential novel marker for prognosis and represents a therapeutic target for the treatment of cervical cancer patients

Clinical evidence has indicated that URG4 is elevated

in patients with certain types of cancer Its expression has been shown to be associated with tumour aggressiveness

in osteosarcomas, prostate cancer and neuroblastomas [11-13] These findings suggest that URG4 may play an important role in cancer development and progression Therefore, we assessed whether it is also clinically asso-ciated with the development and progression of cervical cancer We found that the URG4 mRNA and protein were highly expressed in cervical cancer cell lines and cervical cancer samples (Ib-IIa) We further analysed the relationships between URG4 expression and the clinical characteristics of patients with early-stage cervical cancer There was a significant correlation between URG4 expres-sion and clinical stage, T classification, tumour size, N classification and vaginal involvement, strongly supporting the hypothesis that this protein plays a role in the progres-sion of cervical cancer and may represent a biomarker for the identification of subsets of cervical cancer patients with a more aggressive form of the disease Univariate and multivariate analyses showed that high URG4 expres-sion is a predictor of poor prognosis in these patients Moreover, those with elevated expression showed a 52.5% cumulative OS rate, which was significantly lower than that in the patients with low expression levels (97.1%) Furthermore, the patients (Ib1-Ib2) with high URG4 expression levels had poor outcomes These findings indicate that elevations in URG4 expression may be predictive of a poor prognosis and short survival time for early-stage cervical cancer patients

Table 3 Spearman correlation analysis of URG4 versus

clinicopathological factors

Lymph node metastasis plays a very important role in

the prognosis of cervical cancer patients Recently, a large

number of studies have identified certain genes associated

with cervical lymph node metastasis, including Sam68,

CRAM and EZH2 [7,14,15] Because all of patients were

in early stage in our study, the number of patients with lymph node metastasis was small and they had a good prognosis It was failed to show lymph node metastases was an independent prognostic factor for survival We also showed that URG4 expression was high in the lymph node

Figure 4 Kaplan-Meier curves of univariate analysis data (log-rank test) (a and b) The overall survival (OS) (a) and disease-free survival (DFS) (b) for the patients with high versus low URG4 expression (c) The OS for the patients at stages Ib1-Ib2 with high versus low URG4 expression (d) The OS for the patients without lymph node metastasis with high versus low URG4 expression (e) The OS for the patients with high versus low URG4 expression who received concurrent chemotherapy and radiation therapy (f) The OS for the patients with high versus low URG4 expression who received radiation therapy.

metastasis subgroup and that it was significantly

corre-lated with lymph node metastasis In a more detailed

survival study, we observed a significant correlation

between shorter OS and high URG4 expression in the

“without lymph node metastasis” subgroup This suggests

that URG4 may be a useful prognostic marker for cervical

cancer patients without lymph node metastasis URG4

is regulated by the Akt-mediated phosphorylation of

FOXO3a, which stimulates the cell cycle [8] or alters

cyclin D1 levels by circumventing a cell cycle checkpoint

(G1 to S phase) [9] It is well known that Akt plays a role

in tumour-induced lymphangiogenesis in colorectal

car-cinoma [16] In the case of lymph node metastasis, we

hypothesise that URG4 may regulate the expression of

VEGF-C though the Akt signalling pathway by mediating

lymphangiogenesis in cervical cancer cells Further

func-tional studies are needed to verify these findings to establish

URG4 as a prognostic marker in cervical cancer and to

clarify its role in carcinogenesis and progression

For patients with lymph node metastasis, further

treat-ments are required To date, radical hysterectomy plus

lymphadenectomy or chemoradiation have been the

stand-ard treatments for early-stage cervical cancer patients

[17,18] A prospective, randomised clinical trial has shown

that concurrent chemotherapy and radiotherapy improves

the survival time of early-stage cervical cancer patients

with high-risk prognostic factors compared with RT alone

[19] In our study, the patients with any of the“high-risk”

prognostic factors, including pelvic lymph node metastasis,

positive parametrial involvement, positive surgical margin,

deep stromal invasion and large tumor size (over 4 cm),

re-ceived chemotherapy and radiotherapy The patients who

were only with lymph vascular space invasion or vaginal

involvement received RT However, those who refused

concurrent chemotherapy were treated with RT alone

We found that higher URG4 expression was correlated

with a significantly shorter OS in the concurrent

chemother-apy and radiotherchemother-apy subgroup However, no correlation

was observed in the subgroup receiving only radiotherapy This indicates that URG4 expression is a more signifi-cant predictor of the prognosis of early-stage cervical cancer patients who require concurrent chemotherapy and radiotherapy

Recently, a number of studies have focused on targeted drugs for the treatment of cervical cancer Tewariet al have found that chemotherapy combined with bevacizumab improves OS in recurrent, persistent or metastatic cervical cancer patients [20] Nogueira et al have revealed that EGFR inhibitors combined with chemoradiation result

in high levels of complete remission in patients with lo-cally advanced cervical cancer [21] Consistent with the above findings, our study exploring URG4 as a target of cervical cancer has shown extremely promising results that may be of future clinical value

Conclusions

In conclusion, this is the first study to assess the expression and clinical significance of URG4 in early-stage cervical cancer URG4 could represent a prognostic biomarker and therapeutic target for early-stage cervical cancer patients

Additional file

Additional file 1: Figure S1 Kaplan-Meier curves with univariate analysis (log-rank test) (a) The OS for the patients with stages IIa1-IIa2 cervical cancer and high versus low URG4 expression (b) The OS for the patients with lymph node metastasis and high versus low URG4 expression.

Abbreviations URG4: Upregulator of cell proliferation 4; IHC: Immunohistochemistry; OS: Overall survival; DFS: Disease-free survival; FIGO: International federation

of gynaecology and obstetrics; HCC: Hepatocellular carcinoma;

PBS: Phosphate-buffered saline; RIPA: Radioimmunoprecipitation assay buffer; MOD: Mean optical density; EDTA: Ethylenediaminetetraacetic acid; PVDF: Polyvinylidene fluoride; RT: Radiotherapy; N: Normal cervical epithelial cell line.

Competing interests The authors declare that they have no competing interests.

Table 4 Univariate and multivariate analyses of various prognostic parameters in the patients with cervical cancer Cox regression analysis

*The factors in multivariate analysis included Age, Squamous cell carcinoma antigen, FIGO stage, Tumor size, Histological type, Histological differentiation, Deep stromal invasion, Lymphovascular space involvement, Positive parametrium, Positive surgical margin, Vaginal involvement, N classification, M classification, Concurrent chemotherapy and Radiotherapy, Radiotherapy and Expression of URG4.

(The endpoint is OS).